Standardized Extract And Its Use In The Manufacture Of A Medicament

ABSTRACT

The present invention refers to a medicament which activates gastrointestinal movement. The medicament consists essentially of Cucurbitacin D and one or more substances form the group including Cucurbitacin A, Cucurbitacin B, Cucurbitacin E, Isocucurbitacin B and Isocucurbitacin D. The medicament is made from an extract obtained from Muskmelon Base, the fruit stem of  Cucumis melo  L. The medicament can promote enterokinesia in both normal animals and in animal models where gastrointestinal movement is inhibited. It has also been demonstrated to activate the movements of the stomach, small and large intestines. The extract may be used alone or in combination with the other botanical ingredients or chemical substances to form the pharmaceutical preparation.

TECHNICAL FIELD OF THE INVENTION

The present invention relates a standardized extract, a pharmaceuticalformulation and the extracts use in the manufacture of a medicament fortreating a number of different conditions.

BACKGROUND OF THE INVENTION

After abdominal surgery, normal intestinal movement function may notroutinely recover in 24 hours due to both trauma and anesthesia (causingearly-stage postoperative enteroparalysis or ileus). The intestinalfunction will be affected by one or more of: irritation, effusion ofabdominal cavity, inflammation and anesthesia effect, which causeintestinal distension and synechia. The later is the main cause ofileus.

It has been reported that the incidence rate of intestinal synechia isas high as 60-70% in all abdominal surgery, 5% of which developintestinal ileus. The recurrence rate of ileus is high (over 15%) aftersynechia lysis. Indeed, the more operations, the higher the recurrencerate. [Editor: Chen Qi, Thoughts and Methods in Study on MedicinalEffectiveness of Chinese Medicine, People's Medical Publishing House p.491-492, 2005].

The current treatments for intestinal distension and synechia in theclinic are Neostigmine injection or oral-administration of different“purgative” TCM formulas.

Although Neostigmine injection has good effect, and there are norestraints on its use, it has been reported that it could causesignificant side effects, e.g. intestinal colic, increased glandsecretion and muscle vibration. [Jiang MX and Yang ZC: MedicalPharmacology, People's Medical Publishing House, 3rd ed. Beijing, 1997,p 116].

The alternative, oral-administration of TCM formulas, also has somedisadvantages, e.g. large volumes are difficult to take, and are nottraditionally recommended immediately post operatively (postoperativepatients should be fasted until flatus occurs and flatus is a sign ofrecovering gastrointestinal function). It has however been reported thatthe earlier the use of TCM formula after surgery, the better thetherapeutic results could be. [Ma GX and Hou BZ: Clinical analysis on405 cases of postoperative intestinal distension with Pai Qi Decoction,Chinese Medicine of Factory and Mine, 2002, 15(1): p 72-73].

Kudingxiang (Muskmelon-Base) is the dried fruit stem of Cucumis melo L.(Cucurbitaceae family). The functions and indications of Muskmelon-Base(Kudingxiang) described in the Chinese Materia Medica (Vol. V. pp. 4580)and Inner Mongolia Standard for Materia Medica (1988, pp. 64) are:

Inducing vomiting and diuresis,

Expelling dampness and reducing jaundice.

it is used to treat:

Stroke,

Epilepsy,

Sore throat,

Phlegmatic hygrosis choking,

Shortness of breath,

Dyspepsia,

Abdominal fullness, and

Damp-heat type of jaundice in TCM terms.

It is regarded as an emetic herb in the TCM system due to itsstimulating action upon the gastric mucosa and its reflex triggering ofthe central vomiting nerve.

Cucurbitacins are the main chemical substances in Muskmelon Base. It hasbeen reported in the journal of Chinese Traditional and Herbal Drugs(Vol. 23, No. 11, 1992) that Cucurbitacins have cytotoxicity andanti-tumour activity, offer hepatic protection, anti-hepatitis,improvement of immune function, anti-chemocarcinogenesis, an increase ofcapillary permeability, and induced vomiting.

A product made from Muskmelon Base, and currently sold in the Chinesemarkets, is “Hu Lu Su” tablet (meaning Cucurbitacins tablet). Theproduct specification was recorded in the ‘Standard Specification ofTraditional Chinese Medicines (Vol. 19)’ issued by the Ministry ofHealth, China.

The tablets comprise a purified ethanol extract of the raw material ofMuskmelon Base and the main chemical in the tablet is Cucurbitacin B,the content of which is no less than 60% as stated in the above StandardSpecification. The functions and indications for the tablet include:

Eliminating toxins and clearing heat, and

Inducing diuresis and expelling jaundice

and it is used as an adjunct treatment for persistent hepatitis due tosevere toxic heat, chronic hepatitis and primary hepatic carcinoma.

Muskmelon Base did not have the function of ‘purgation’ in TCM but Ederyet al, reported that intravenous injection of Cucurbitacin D isolatedfrom Ecballium elaterium L. induced diarrhea in conscious cats and dogs,and also stimulated enterokinesia in anesthetized dogs. But on theisolated guinea pig ileum, a concentration of 54 mg/ml Cucurbitacin Ddid not cause any visible effect. [H. Edery, G. Schatzberg Porath, S.Gitter, Pharmacodynamic activity of Elatericin (Cucurbitacin D) Arch IntPharmacodyn. 1961, 13(3˜4):315˜335].

It is an aim of the present invention to develop a medicament which willprevent intestinal distension and synechia and speed upgastroenterokinesia restoration. Such a medicament will have asignificant impact on a patient's recovery after surgery.

The applicant has developed a standardized extract comprisingCucurbitacin D from Cucumis melo L and used this to develop aformulation for rectal delivery which has proved effective in animalstudies.

The extract may be used alone or in combination with the other botanicalingredients or chemical substances to form the pharmaceuticalpreparation.

Furthermore the formulation avoids the hepatic by-pass effect associatedwith oral forms, is quick acting and would appear to benefit from noobvious muscle spasm side effect, a problem with Neostigmine the currentpreferred treatment.

TECHNICAL FIELD OF THE INVENTION

According to a first aspect of the present invention there is provided astandardized extract of Cucumis melo L.

The extract is standardized by reference to the content of one or moremembers of the family of compounds known as Cucurbitacins (including theIsocueurbitacins).

These include Cucurbitacin D, Cucurbitacin A, Cucurbitacin B,Cucurbitacin E, Isocucurbitacin B and Isocucurbitacin D.

Preferably the extract is standardized with reference to Cucurbitacin Dwhich is believed to be an active ingredient.

However, the extract may alternatively be standardized with reference toCucurbitacin B.

Indeed, the extract may be standardized by reference to an amount/rangeof both Cucurbitacin D and Cucurbitacin B.

Preferably the extract is a whole extract of Cucurbitacins.

Alternatively, the extract may be a purified or a selective fraction.

The extract or a selective fraction preferably comprises at leastCucurbitacin D and more preferably additionally Cucurbitacin B.

Indeed, it could comprise, without limitation, any permutation of thetwo, three, four, five or six specified Cucurbitacins andIsocucurbitacins.

Most preferably the extract comprises the Cucurbitacins B and D plus atleast one

of the following: Cucurbitacin A, Cucurbitacin E, Isocucurbitacin B andIsocucurbitacin D.

Even more preferably the extract comprises the Cucurbitacins B and Dtogether with each of the following: Cucurbitacin A, Cucurbitacin E, andoptionally also, each of Isocucurbitacin B and or Isocucurbitacin D.

It will be apparent from the above that in order to arrive at aconsistent extract it is important to standardize the dose against oneor more markers/perceived actives.

In a favored embodiment the ratio of Curcurbitacin B/Curcurbitacin D inthe extract is from between 30:1 to 1:10, more preferably still between10:1 and 1:2 and most preferably still between 3:1 and 1.1.

Since the applicant has demonstrated that extracts containing bothCucurbitacin D and Cucurbitacin B demonstrate activity, it is preferredthat one or more of these compounds are used to standardize the extractfor use as a medicament. Indeed standardizing the extract againstCucurbitacin D or B content or within a closely defined range may bepreferred.

Extracts according to the invention can be obtained by a number ofalternative extraction methodologies which may additionally includefurther “purification” or additional extraction steps.

By way of example only, suitable solvent extraction methods include, butare not limited to, the use of water, polar solvents, such as ethanoland methanol, and their aqueous solutions.

The extraction methods, using the solvents mentioned above, include heatreflux extraction (including soxhlet extraction), percolation andmaceration at room temperature.

The preferred solvent is water.

The preferred extraction method is a heat reflux extraction.

Based on the physical property of the raw material and the quantity ofwater used, the extraction process may be repeated up to four times. Theusual extraction duration is 0.5-2 hours and the preferred extractionduration is one hour. The water quantity to be used is 8-10 folds of theraw material weight.

Preferably, the extraction is followed by a purification step. Thepreferred purification step is an ethanol precipitation step.

When the first concentrate is added to the ethanol solution the finalethanol concentration should be between 50% and 80%, preferably 70%.Under this condition, the content of the active compounds andcomposition are most suitable for the pharmaceutical application.

Thus, a preferred combined extraction and purification process maycomprise the steps of:

-   -   Pulverizing the raw material of Muskmelon Base,    -   Extracting with water and obtaining a liquid extract,    -   Concentrating the liquid extract to obtain a first concentrate,    -   Adding the first concentrate to ethanol,    -   Stir thoroughly, set aside, filter and concentrate the solution        to obtain a second concentrate,    -   Recover the ethanol and dry the second concentrate to obtain a        purified extract.

Alternative or additional purification processes include liquid-liquidpurification and resin purification.

Thus, the first and second concentrates of for example, the preferredcombined extraction purification process outlined above may additionallybe purified by liquid-liquid partition. Suitable solvents includechloroform, methylene chloride, ether, and ethyl acetate.

A liquid-liquid partition step should usually be carried out a pluralityof times, say 2-5 times, preferably 3 times.

After the liquid-liquid partition, the solvents are recovered. Theconcentrates from the liquid-liquid partition are dried to obtainextracts which can be used in the manufacture of a medicament.

An alternative purification method is to dissolve the first or secondconcentrates in water, and place the solution onto a column filled witha macro-porous resin. The process may comprise the steps of

-   -   Wash the column with water,    -   Allow it to run through the column,    -   Throw the water solution,    -   Elute with one, or several, of the following solvents: methanol,        ethanol, aqueous ethanol and aqueous methanol,    -   Collect the eluents,    -   Concentrate the eluent under vacuum and recover the solvent, and    -   Dry the concentrates to obtain a purified extract.

The macro-porous absorption resin can be D 101, AB-8 or any othersuitable resin.

D-101 is manufactured by Huishi Resin Factory, Shanghai, China. It is anon-polar resin with the following specification:

TABLE 1 % Water 65-75 Wet density g/ml 0.65-0.75 Particle size(0.25-0.84 mm) > or = to 95 Surface area rate m²g 500-550 Averagechamber diameter  90-100

AB-8 is manufactured by Nankai Chemicals, Tianjin, China. It is anon-polar resin with the following specification:

TABLE 2 % Water 65-75 Wet density g/ml 1.00-1.10 Particle size(0.25-0.84 mm) > or = to 95 Surface area rate m²g  480-5250 Averagechamber diameter 13-14

The elution can be an isocratic or gradient elution.

The drying methods used in the preparation of the extracts may include,but are not limited to, vacuum drying, and spray drying.

According to a second aspect of the present invention there is provideda pharmaceutical formulation comprising a standardized extract ofCucumis melo L in unit dosage form.

Preferably the pharmaceutical formulation is standardized with referenceto a Curcurbitacin (including isocurcurbitacins).

Most preferably the standardizing Curcurbitacin is Curcurbitacin D,although Curcurbitacin B may be used.

Preferably, where the ratio of Cucurbitacin B/Cucurbitacin D is between3:1 and 1:1, the formulation is standardized to provide a daily dose(based on a 65 kg patient) containing from 0.0040 mg to 40 mg ofCucurbitacin D, more preferably from 0.04 mg to 4.0 mg of Cucurbitacin Dand most preferably still between 0.1 mg and 1.6 mg, with the preferredstandard dose containing about 0.40 mg.

Alternatively, where the ratio of Cucurbitacin B/Cucurbitacin D isbetween 3:1 and 1:1, the formulation may be standardized to provide adaily dose (based on a 65 g patient) containing from 0.0064 to 64 mgCucurbitacin B, more preferably from 0.064 to 6.4 mg Cucurbitacin B andmost preferably still between 0.16 mg and 2.6 mg, with the preferredstandard dose containing about 0.64 mg.

Preferably the pharmaceutical formulation is suitable for rectaldelivery. Most preferably it takes the form of a suppository.

The carriers for rectal administration include:

-   -   Lipopholic bases including: natural fatty acid esters,        semi-synthetic glycerides or hydrogenated vegetable oil;    -   Hydropholic bases including: gelatin glycerin, polyethylene        glycol, polyoxyl [40]Stearate, Tween 61, polyoxethylene sorbitan        monostearate or poloxamer (Pluronics);    -   Additives including: surfactants such as Tween 80;    -   Chelators including: EDTA, trisodium citrate dehydrate or        enamine derivatives;    -   Nonsteroidal anti-inflammatory agents including: sodium        salicylate;    -   Antioxidants including: Butylated hydroxyanisole (BHA),        Butylated hydroxytoluene (BHT) or Gallocatechin gallate; and    -   Preservatives including para-hydroxybenzoic acid esters.

The medicament administrated by a rectal route is absorbed through therectal mucosa and consequently about 50% to 70% of the medication doesnot go into the liver. As a result metabolism of the medication in theliver, and liver toxicity, are reduced. The rectal administration alsoavoids irritation to the gastric mucosa.

A rectal suppository is easy to use and it can be given to in-patientsas well as out-patients. It is easily absorbed, fast to take effect andhas high bioavailability. The medicament can shorten the duration of therecovery and rehabilitation post operatively with no limitation on thesubject's gastrointestinal function.

However, alternative modes of delivery are contemplated and within thescope of the invention.

Thus, the extracts can be made into different dosage forms byconventional pharmaceutical preparation. The extract of the inventionmay be combined with any physiological acceptable drug carrier to form apharmaceutical preparation in order to provide the medication to thesubjects through different routes, for example, oral or via injection.Injectable forms include subcutaneous injection, intradermal injection,intramuscular injection, intraperitoneal injection, intravenousinjection, intranasal injection and acupoint injection. Alternativelythe medicament might be introduced directly at the surgical site.

The extract in the present invention could be made into injectablemedications including solutions, emulsions, suspensions and injectablepowders. As injection formulations, physiological acceptable carriersinclude pharmaceutically acceptable medium and additives may be used.The pharmaceutically acceptable mediums include water, non-aqueoussolvents, or compound solvents. The non-aqueous solvents includeinjectable oils, ethanol, glycerin, propylene glycol, polyethyleneglycol, benzyl benzoate, dimethyl acetamide, dimethyl sulfoxide, andisopropyl myristate. The additives include solubilizers, antioxidants,isotonic regulator, buffer, suspending agents, stabilizers, chelatingagent, antibacterial agent, and tiller, such as Tween-80, phosphate,methyl cellulose, creatinine, sodium sulfite, sodium choiride, ethylenediamine tetra-acetic acid disodium salt, lactose, and phenol.

Based on significant pharmacological studies, the applicant has foundthat a Muskmelon-Base extract made from, for example, aqueous extractionand ethanol precipitation possesses significant beneficial effectsincluding the following:

Activating gastroenterokinesia;

Stimulating gastrointestinal movement in non-surgery

Impelling the large and small intestines;

Activating gastric emptying; and

Shortening the excretion intervals and increasing the quantity offaeces.

According to a third aspect of the present invention there is provided astandardized extract of Cucumis melo L for use in the manufacture of amedicament for the treatment of, or to effect, one or more of thefollowing conditions:

Activating gastroenterokinesia;

Postoperative gastroenterokinesia restoration;

Shortening the duration of postoperative flatus;

Treatments of constipation;

Treating postoperative intestinal adhesion;

Treating Intestinal adhesion and ileus;

Stimulating gastrointestinal movement in non-surgery;

Impelling the large and small intestines;

Activating gastric emptying;

Smoothes bowel movement; and

Shortening excretion intervals and increasing the quantity of faeces.

It will be apparent from the above that in order to arrive at aconsistent extract it is important to standardize the dose against oneor more markers/perceived actives.

Since the applicant has demonstrated that extracts containing bothCucurbitacin D and B demonstrate activity, it is preferred that one ormore of these compounds are used to standardize the extract for use as amedicament.

The preferred marker is Curcurbitacin D although Curcurbitacin B oranother Cucurbitacin or Isocucurbitacin may be used.

In a favored embodiment the ratio of Curcurbitacin B/Curcurbitacin D inthe extract is from between 30:1 to 1:10, more preferably still between10:1 and 1:2 and most preferably still between 3:1 and 1.1.

Preferably the amount of Curcubitacin D present in the total weight ofCurcubitacins, including isocurcubitacins, comprises from 5% to 50%,more preferably 8% to 41%.

More particularly, the applicant has demonstrated that:

-   -   1. After a single dose rectal administration of the medicament,        the proximal colon constriction of anaesthetised rats was        significantly improved within 30 minutes, and it reached a peak        between 30 min. and 60 min.    -   2. The medicament of the present invention can significantly        activate the intestinal impellent of the mice with intestinal        mechanical injury. At a high dose its function was equivalent to        Neostigmine, but was without muscle spasm, a common side effect        found with Neostigmine.    -   3. The medicament can additionally:        -   Activate the intestinal impellent of the animals with            gastrointestinal hypo-function induced by atropine,        -   Shorten the excretion intervals,        -   Increase the quantity of faeces, (count for 4 hours) and        -   Improve the function of gastric emptying in the animals with            gastrointestinal hypo-function induced by atropine.

As stated above, the medicament of the present invention can promotegastroenterokinesia in both normal and inhibited gastrointestinalanimals and also promote movements in the stomach, small and largeintestines. Comparatively speaking the strength of the effect is inorder: large intestine>small intestine>stomach.

The Muskmelon-Base extract possesses the excellent features of:

A small dosage quantity,

A low frequency of administration (potentially single dose), and

A fast onset of action.

Practically, the Muskmelon Base extract can activategastroenterokinesia, and based on its characteristics, can be used inthe treatment or prevention of

-   -   Constipation,    -   Intestinal adhesion and ileus, and    -   Promotion of flatus in order to activate gastroenterokinesia        restoration after abdominal surgery.

According to a forth aspect of the invention there is provided a methodof treatment of the human or animal body comprising administering anextract or pharmaceutical formulation of the invention to a patient.

The present invention will be further described, by way of Example only,with reference to the following formulation and data in which:

FIG. 1 a shows the chemical structure of Cucurbitacin D;

FIG. 1 b shows the chemical structure of Cucurbitacin B;

FIG. 1 c shows the chemical structure of Cucurbitacin E;

FIG. 1 d shows the chemical structure of Isocucurbitacin B;

FIG. 1 e shows the chemical structure of Cucurbitacin A;

FIG. 1 f shows the chemical structure of Isocucurbitacin D;

FIG. 2 a is an HPLC fingerprint of the first concentrate used in Example1;

FIG. 2 b is an HPLC fingerprint of the Muskmelon Base extract used inExample 2;

FIG. 2 c is an HPLC fingerprint of the second concentrate used inExample 1;

FIG. 2 d is an HPLC fingerprint of the chloroform purified aqueousextract used in Example 8;

FIG. 2 e is an HPLC fingerprint of the extract treated by macro-porousresin as used in Example 11;

FIG. 3 is a graph showing the impelling percentage of the smallintestine in normal mice;

FIG. 4 is a graph showing the impelling percentage of large intestine innormal rats;

FIG. 5 is a graph showing the gastric emptying percentage in normalmice;

FIG. 6 is a graph showing the intestinal impelling percentage in micewith mechanical injury;

FIG. 7 is a graph showing the Intestinal impelling percentage in micewith gastrointestinal hypodynamia caused by atropine;

FIG. 8 is a graph showing the number of contractions—effect on proximalcolon contraction in anesthetized rats;

FIG. 9 is a graph showing the rate of contractions—effect on proximalcolon contraction in anesthetized rats;

FIG. 10 is a graph showing the amplitude indices—effect on proximalcolon contraction in anesthetized rats;

FIG. 11 is a graph showing the rate of amplitude—effect on proximalcolon contraction in anesthetized rats;

FIG. 12 is a graph showing the gastro emptying test—gastrointestinalhypodynamia in a mouse model caused by atropine; and

FIG. 13 is a graph showing the recorded ex vivo study result—effect of60 mg/20 ml dose on isolated guinea pig ileum (From the top to thebottom, the recorded waves are: before administration, 0, 2, 5, and 10minutes after administration).

In each of the HPLC fingerprints (FIGS. 2 a-2 e):

Peak 1 is Cucurbitacin A;

Peak 2 is Cucurbitacin D;

Peak 3 is Isocucurbitacin D;

Peak 4 is Cucurbitacin B;

Peak 5 is Cucurbitacin E; and

Peak 6 is Isocucurbitacin B.

The chromatographic conditions were as follows:

-   -   Column: DIKMA Diamonsil HPLC Column C18 250 mm×4.6 mm, 5 μm    -   Column Temperature: 30° C.    -   Wavelength: 232 nm    -   Flow rate: 1.0 min/nl    -   Mobile phase: A: acetonitrile: tetrahydrofuran (1:1);    -   B: gradient elution with two-phase of 0.1% phosphoric acid—water        (0 min: 30% A; 40 min: 70% A)

DETAILED DESCRIPTION

The detailed description given below is set out in three parts.

-   -   Examples 1 to 13 describe different methods of preparing        extracts (extraction and purification) containing Cucurbitacins        and the resulting extracts;    -   Examples 14 to 18 describe formulations made from a number of        these extracts; and    -   Experiments 1 to 12 provide details of the studies conducted        which provide credible support for the medical indications        claimed.

Part 1. Extracts and Extraction/Purification Methodology

The following examples illustrate a variety of methodologies that can beused to obtain Cucurbitacin containing extracts for use in medicine.

Example 1 Aqueous Extraction and Ethanolic Precipitation

The raw material of Muskmelon Base (5 kg) was pulverised into a coarsepowder and subjected to the following regime:

-   -   1. Add 50 kg of water to the coarse powder and boil for        approximately 2 hours;    -   2. Decant the solution;    -   3. Add a further 40 kg of water to the residue and boil for a        further 1 hour, then decant the solution;    -   4. Add a further 40 kg of water to the residue and boil for a        further 1 hour, then decant the solution;    -   5. Collect the three solutions and filter;    -   6. Concentrate the solution to obtain a first concentrate (7.5        kg);    -   7. Add ethanol to the concentrate to get a 70% ethanol solution.        Agitate fully, set aside, allow to precipitate for 24 hours and        filter;    -   8. Recover the ethanol to obtain a second concentrate;    -   9. Spray-dry the second concentrate to obtain a solid extract.

The solid extract obtained had a composition as shown in Table 3 below:

TABLE 3 Cucurbitacin content % (by wt of extract) Cucurbitacin B 0.99Cucurbitacin D 0.61 Cucurbitacin E 0.14 Cucurbitacin A 0.12Isocucurbitacin D 0.09 Isocucurbitacin B 0.06 TOTAL Ratio ofCucurbitacin B to 1.62:1.0 Cucurbitacin D Ratio of Cucurbitacin D to0.30:1.0 Total Cucurbitacins (including isocucurbitacins)

Example 2 Solvent Extraction Using Methanol

The raw material of Muskmelon Base (5 kg) was pulverised into a coarsepowder and subjected to the following regime:

1. Add 25 kg of methanol and reflux extract with a Soxhlet for 6 hours;

2. Concentrate the liquid extract under vacuum and spray dry to obtain asolid extract.

The solid extract obtained had a composition as shown in Table 4 below:

TABLE 4 Cucurbitacin content % (by wt of extract) Cucurbitacin B 5.83Cucurbitacin D 0.63 Cucurbitacin E 0.03 Cucurbitacin A 0.36Isocucurbitacin D Not detected (<0.01%) Isocucurbitacin B 0.76 TOTALRatio of Cucurbitacin B to 9.25:1.0 Cucurbitacin D Ratio of CucurbitacinD to 0.08:1.0 Total Cucurbitacins (including isocucurbitacins)

Example 3 Aqueous Extraction and Ethanolic Precipitation (2)

The raw material Muskmelon Base (5 kg) was pulverised into a coarsepowder and subjected to the following regime:

-   -   1. Add 30 kg of water and boil for 30 minutes,    -   2. Pour off the solution,    -   3. Repeat step 1 a further three times,    -   4. Pool the solutions and filter,    -   5. Concentrate the solution to obtain a first concentrate (2.5        kg),    -   6. Add ethanol to form a 65% ethanol solution, agitate fully and        set aside to precipitate for 18 hours,    -   7. Filter, and recover the ethanol under vacuum to obtain a        second concentrate,    -   8. Dry the concentrate under vacuum to obtain a solid extract.

The solid extract obtained had a composition as shown in Table 5 below:

TABLE 5 Cucurbitacin content % (by wt of extract) Cucurbitacin B 0.87Cucurbitacin D 0.51 Cucurbitacin E 0.11 Cucurbitacin A 0.10Isocucurbitacin D 0.07 Isocucurbitacin B  0.05. TOTAL Ratio ofCucurbitacin B to 1.71:1.0 Cucurbitacin D Ratio of Cucurbitacin D to0.30:1.0 Total Cucurbitacins (including isocucurbitacins)

Example 4 Ethanolic Extraction

The raw material of Muskmelon Base (4 kg) was pulverised into a coarsepowder and subjected to the following regime:

-   -   1. Add 25 kg of 75% ethanol and subject it to reflux extraction,        with heat, for 4 hours to obtain a liquid extract;    -   2. Spray-dry the extract to obtain the solid extract.

The solid extract obtained had a composition as shown in Table 6 below:

TABLE 6 Cucurbitacin content % (by wt of extract) Cucurbitacin B 2.91Cucurbitacin D 0.58 Cucurbitacin E Not detected (<0.01%) Cucurbitacin A 0.18. Isocucurbitacin D Not detected (<0.01%) Isoeucurbitacin B 0.36TOTAL Ratio of Cucurbitacin B to 5.02:1.0 Cucurbitacin D Ratio ofCucurbitacin D to 0.14:1.0 Total Cucurbitacins (includingisocucurbitacins)

Example 5 Aqueous Extraction and Ethanolic Precipitation (3)

The raw material of Muskmelon Base (3 kg) was pulverized into a coarsepowder and subjected to the following methodology.

-   -   1. Add 30 kg of water and boil for one hour,    -   2. Pour off the solution,    -   3. Repeat step 1 and 2 a further two times,    -   4. Collect the solutions, filter and concentrate to obtain a        first concentrate (5 kg),    -   5. Add ethanol to form a 75% ethanol solution, agitate fully and        set aside for precipitation for 30 hours,    -   6. Filter and recover the ethanol under vacuum to obtain a        second concentrate,    -   7. Dry the concentrate under vacuum to obtain a solid extract.

The solid extract obtained had a composition as shown in Table 7 below:

TABLE 7 Cucurbitacin content % (by wt of extract) Cucurbitacin B 0.81Cucurbitacin D 0.48 Cucurbitacin E 0.10 Cucurbitacin A 0.10Isocucurbitacin D 0.06 Isocucurbitacin B 0.04 TOTAL Ratio ofCucurbitacin B to 1.69:1.0 Cucurbitacin D Ratio of Cucurbitacin D to0.30:1.0 Total Cucurbitacins (including isocucurbitacins)

Example 6 Aqueous Extraction and Ethanol Precipitation (4)

The raw material of Muskmelon Base (4.5 kg) was pulverised into a coarsepowder and subjected to the following methodology.

-   -   1. Add 35 kg water and boil it for one hour,    -   2. Pour off the solution,    -   3. Repeat a further 3 times,    -   4. Pool the solutions, filter and concentrate to obtain a first        concentrates (54),    -   5. Add ethanol to form an 80% ethanol solution, agitate fully        and set aside for precipitation for 30 hours,    -   6. Filter and recover the ethanol under vacuum to obtain a        second concentrate,    -   7. Spray-dry the concentrates to obtain the solid extract.

The solid extract obtained had a composition as shown in Table 8 below:

TABLE 8 Cucurbitacin content % (by wt of extract) Cucurbitacin B 0.62Cucurbitacin D 0.58 Cucurbitacin E 0.12 Cucurbitacin A 0.10Isocucurbitacin D 0.05 Isocucurbitacin B 0.06 TOTAL Ratio ofCucurbitacin B to 1.07:1.0 Cucurbitacin D Ratio of Cucurbitacin D to0.38:1.0 Total Cucurbitacins (including isocucurbitacins)

Example 7 Ethanolic Extraction (2)

The raw material Muskmelon Base (5 kg) was pulverised into a coarsepowder and subjected to the following methodology:

-   -   1. Add an adequate amount of 50% ethanol to cover the powder and        soak at room temperature over night,    -   2. Apply a percolation method using a further 60 kg of 50%        ethanol solution,    -   3. Concentrate the solution under vacuum,    -   4. Spray dry to obtain a solid extract.

The solid extract obtained had a composition as shown in Table 9 below:

TABLE 9 Cucurbitacin content % (by wt of extract) Cucurbitacin B 2.78Cucurbitacin D 0.42 Cucurbitacin E Not detected (<0.01%) Cucurbitacin ANot detected (<0.01%) Isocucurbitacin D Not detected (<0.01%)Isocucurbitacin B 0.03 TOTAL Ratio of Cucurbitacin B to 6.62:1.0Cucurbitacin D Ratio of Cucurbitacin D to 0.13:1.0 Total Cucurbitacins(including isocucurbitacins)

Example 8 Chloroform Purification

The first concentrate, as described in Example 1, was subjected topurification with chloroform as follows:

-   -   1. Dissolve the first concentrate in chloroform (⅔ in volume),    -   2. Separate the chloroform solution,    -   3. Repeat steps 1 and 2 a further two times,    -   4. Collect the combined chloroform solutions, recovery the        chloroform and dry the residue under vacuum to obtain a solid        extract.

The solid extract obtained had a composition as shown in Table 10 below:

TABLE 10 Cucurbitacin content % (by wt of extract) Cucurbitacin B 30.68Cucurbitacin D 24.95 Cucurbitacin E 4.24 Cucurbitacin A 3.50Isocucurbitacin D 4.24 Isocucurbitacin B 3.50 TOTAL Ratio ofCucurbitacin B to 1.23:1.0 Cucurbitacin D Ratio of Cucurbitacin D to0.35:1.0 Total Cucurbitacins (including isocucurbitacins)

Example 9 Liquid/Liquid Purification

The liquid extract, as described in Example 2, was taken and themethanol recovered under vacuum as described below.

-   -   1. Water was added to the extract (same weight as raw materials)        to dissolve the residue and then ether added (the same volume as        water) to perform liquid-liquid partition,    -   2. The ether fraction was recovered and step 1 repeated once        again,    -   3. The ether solutions were combined and the ether recovered,    -   4. The residue was dried under vacuum to obtain a solid extract.

The solid extract obtained had a composition as shown in Table 11 below:

TABLE 11 Cucurbitacin content % (by wt of extract) Cucurbitacin B 65.79Cucurbitacin D 8.45 Cucurbitacin E 0.42 Cucurbitacin A 5.04Isocucurbitacin D 0.35 Isocucurbitacin B 9.4 TOTAL Ratio of CucurbitacinB to 7.79:1.0 Cucurbitacin D Ratio of Cucurbitacin D to 0.09:1.0 TotalCucurbitacins (including isocucurbitacins

Example 10 Liquid/Liquid Purification (2)

The second concentrate as described in Example 5 was taken and furtherpurified as follows:

-   -   1. The concentrate was dissolved with water (same weight as of        raw material),    -   2. Liquid-liquid partition was performed with ½ volumes of ethyl        acetate 5 times,    -   3. The ethyl acetate solutions were pooled and ethyl acetate        recovered,    -   4. The residue was dried under vacuum to obtain a solid extract.

The solid extract obtained had a composition as shown in Table 12 below:

TABLE 12 Cucurbitacin content % (by wt of extract) Cucurbitacin B 18.94Cucurbitacin D 12.50 Cucurbitacin E 2.53 Cucurbitacin A 1.98Isocucurbitacin D 1.84 Isocucurbitacin B 2.02 TOTAL Ratio ofCucurbitacin B to 1.52:1.0 Cucurbitacin D Ratio of Cucurbitacin D to0.31:1.0 Total Cucurbitacins (including isocucurbitacins

Example 11 Resin Purification

The first concentrate as described in Example 1, was diluted with waterand placed onto a column filled with a water pre-balanced macro-porousresin D101 and purified as follows.

-   -   1. The column was eluted with water and then with 90% ethanol;    -   2. The ethanol solution was collected and the ethanol recovered        under vacuum;    -   3. The residue was spray-dried to obtain a solid powder.

The solid extract obtained had a composition as shown in Table 13 below:

TABLE 13 Cucurbitacin content % (by wt of extract) Cucurbitacin B 47.52Cucurbitacin D 17.60 Cucurbitacin E 1.72 Cucurbitacin A 3.01Isocucurbitacin D 1.15 Isocucurbitacin B 3.72 TOTAL Ratio ofCucurbitacin B to 2.70:1.0 Cucurbitacin D Ratio of Cucurbitacin D to0.24:1.0 Total Cucurbitacins (including isocucurbitacins)

Example 12 Resin Purification (2)

The second concentrate as described in Example 6, was diluted with waterand placed onto a column filled with water pre-balanced macro-porousresin AB-8 and purified as follows.

-   -   1. Gradient elution was performed with water then 10%, 50% and        90% ethanol, respectively,    -   2. The water and 10% ethanol solution were thrown and the 50%        and 90% ethanol solutions collected,    -   3. The ethanol was recovered under vacuum to obtain A solid        extract.

The solid extract obtained had a composition as shown in Table 13 below:

TABLE 13 Cucurbitacin content % (by wt of extract) Cucurbitacin B 13.55Cucurbitacin D 13.42 Cucurbitacin E 1.10 Cucurbitacin A 2.97Isocucurbitacin D 0.97 Isocucurbitacin B 0.80 TOTAL Ratio ofCucurbitacin B to 1.01:1.0 Cucurbitacin D Ratio of Cucurbitacin D to0.41:1.0 Total Cucurbitacins (including isocucurbitacins

Example 13 Resin Purification (3)

The concentrate as described in Example 7 was diluted with water andplaced onto a column filled with water pre-balanced macro-porous resinAB-8 and purified as follows.

-   -   1. Gradient elution was performed with water then 10%, 50% and        90% ethanol, respectively,    -   2. The water and 10% ethanol solution were thrown and the 50%        and 90% ethanol solutions collected,    -   3. The ethanol was recovered under vacuum to obtain the solid        extract.

The solid extract obtained had a composition as shown in Table 14 below:

TABLE 14 Cucurbitacin content % (by wt of extract) Cucurbitacin B 36.88Cucurbitacin D 6.25 Cucurbitacin E Cucurbitacin A 0.14 Isoeucurbitacin DIsocucurbitacin B 1.11 TOTAL Ratio of Cucurbitacin B to 5.90:1.0Cucurbitacin D Ratio of Cucurbitacin D to 0.14:1.0 Total Cucurbitacins(including isocucurbitacins)

Part 2. Production of Formulated Medicaments from Extracts Example 14Formulation for Rectal Delivery

A rectal formulation was made as follows:

-   -   1. The solid extract as described in Example 5 was ground into a        fine powder,    -   2. An amount of semi-synthetic glycerides suitable to form unit        dosage (1.2 g) suppositories containing a desired dose (90 mg of        extract) were taken and melted in a water bath at 40° C.,    -   3. The solution and powder were mixed and stirred thoroughly,    -   4. The resulting solution was poured into a mould and cooled.

The resulting rectal suppository contained 90 mg extract standardisedagainst either Cucurbitacin D 0.43 mg and/or Cucurbitacin B 0.73 mg

The product has a ratio of Cucurbitacin B:Cucurbitacin D of 1.69:1.0

Example 15 Formulation for Rectal Delivery (2)

A rectal formulation was made as follows:

-   -   1. The solid extract as described in Example 1 was ground into a        fine powder,    -   2. An adequate amount of 50% ethanol and Tween-80 was added and        the mix agitated fully,    -   3. An amount of semi-synthetic glycerides suitable to form unit        dosage suppositories (1.5 g) containing a desired dose of        extract (60 mg) were taken and melted in a water bath at 40° C.,    -   4. The solution and powder were mixed and stirred thoroughly,    -   5. The resulting solution was poured into a mould and cooled.

The resulting rectal suppository contained 60 mg extract standardisedagainst either Cucurbitacin D 0.37 mg and/or Cucurbitacin B 0.59 mg.

The product has a ratio of Cucurbitacin B:Cucurbitacin D of 1.62:1.0

Example 16 Formulation for Rectal Delivery (3)

A rectal formulation was made as follows:

-   -   1. The solid extract as described in Example 3 was ground into a        fine powder,    -   2. An adequate amount of cocoa butter and poloxamer (Pluronics)        were melted in a water bath at 40° C.,    -   3. The solution was mixed with the powder and stirred        thoroughly,    -   4. The solution was poured into a mould and allowed to cool.

The resulting rectal suppository (1.5 g) contained 20 mg extractstandardised against either Cucurbitacin D 0.10 mg and/or Cucurbitacin B0.17 mg.

The product has a ratio of Cucurbitacin B:Cucurbitacin D of 1.71:1.0

Example 17 Formulation for Rectal Delivery (4)

A rectal formulation was made as follows:

-   -   1. Take the solid extract as described in Example 8 and grind        into a fine powder,    -   2. Take an adequate amount of semi-synthetic glycerides and        poloxamer (Pluronics) and melt them in a water bath at 40° C.,    -   3. Mix the solution with the powder and stir thoroughly,    -   4. Pour the solution into a mould and cool it down.

The resulting rectal suppository (1.2 g) contained 15 mg extractstandardised against either Cucurbitacin D 3.74 mg and/or Cucurbitacin B4.60 mg.

The product has a ratio of Cucurbitacin B:Cucurbitacin D of 1.23:1.0.

Example 18 Formulation for Rectal Delivery (5)

A rectal formulation was made as follows:

-   -   1. Take the solid extract as described in Example 6 and powder        it,    -   2. Melt an adequate amount of spiceleaf kernal oil (Lindera        communis Hemsl) and Tween −80 at 40° C. in a water bath,    -   3. Add the powder into the solution and stir thoroughly,    -   4. Pour the mixture into the mould and cool it down.

The resulting rectal suppository (1.0 g) contained 50 mg extractstandardised against either Cucurbitacin D 0.29 mg and/or Cucurbitacin B0.31 mg.

The product has a ratio of Cucurbitacin B:Cucurbitacin D of 1.07:1.0

Part 3—Evidence for Claimed Activity

In the following section a solid extract as described in Example 1 wasused and for the convenience of understanding, the relationship ratio Kof the daily dose per kg body weight between different species is givenin the table below.

human rat rabbit mice K 0.11 0.71 0.37 1

Experiment 1—Maximum Tolerable Dose Test

This experiment was conducted to determine the maximum tolerable dose(MTD), by rectal administration, of an extract of the type described inexamples 1.

In this experiment, 10 male and 10 female SD rats weighting 200-240 gwere used. They were fasted for 20 hours before the experiment. The ratswere given rectal administrations of the Muskmelon-Base extract (dilutedwith saline to a concentration of 40%) at the dose of 153 mg/kg/d. Thereactions of the rats, including behavior, limb movement, intake ofwater and food, urine and stool were recorded. The investigation lastedfor 14 continuous days.

Most of the animals produced watery feces with mucus between 30-120mins. after medication, and normal feces 6-8 hours after medication.Some animals curled up with little movement at the early stage followingmedication but returned to normal activity after 30 mins. All theanimals grew normally with no death occurring in a 14-day observationperiod. There was no obvious sign of toxicity and no organ abnormalitywas found.

The maximum dose of the extract, by rectal administration, was 153mg/kg/day which is over 200 times the recommended human clinic dose. Itindicated that the extract has a good safety profile when administeredas a single dose and is a guide for safe clinical use.

Experiment 2—Rectal Mucosal Irritation Test

The solid extract as described in Example 1 was continuously given torabbits at both high and low dose for 7 days.

The animals at low dose (4.64 mg/kg) showed no obvious sign ofirritation to the rectal mucosa and this dose was equivalent to 7.14times the recommended human clinical dose of 0.65 mg.

The animals at the high dose (18.55 mg/kg) showed a slight irritation tothe rectal mucosa but it was reversible and this dose was equivalent to28.5 times the recommended human clinical dose of 0.65 mg.

The experimental result suggested that the extract would be non-irritantwhen administered to humans as a single dose of 0.65 mg/kg/d.

Experiment 3—Excretion Promotion Study

In this experiment, 50 male ICR mice weighing 23-27 g respectively weredivided randomly into 5 groups with 10 per group, i.e. normal control,positive control and three dose groups at 2.48 mg/kg, 4.96 mg/kg and9.92 mg/kg, respectively. They were fasted for 24 hours before theexperiment.

The mice in the positive control group were given an injection of 0.1ml/10 g body weight of Neostigmine hypodermically while the mice in thenormal control groups were given rectal administrations of saline of0.02 ml/10 g body weight. The mice in the three dose groups were givenrectal administrations of the Muskmelon-Base extract (diluted withsaline to give desired doses and given 0.02 ml/10 g body weight).

After medication, all the mice were intra-gastrically given infusions ofIndia ink (10% ink, diluted with saline, at the dose 0.02 ml/10 g ofbody weight). The mice were individually put into a cage with whitefilter paper flooring. The time of excretion and quantity of the feceswere recorded during 4-hour observation periods.

By comparison with the normal control group, and by use of statisticalanalysis, the duration for the first excretion of black feces of themice in the positive control group and three dose groups were shortenedby 53%, 34%, 39% and 49% (p<0.01), respectively. The quantity of thefeces from the positive control group and three dose groups wereincreased by 49% (p<0.01), 19% (p<0.05), 29% (p<0.01) and 49% (p<0.01),respectively.

The results suggested that the extracts at all three doses could clearlypromote excretion in normal mice and the effect was dose dependent.

Experiment 4—Study on Small Intestine Impelling

In this experiment, 50 male ICR mice weighting 23-27 g respectively weredivided randomly into 5 groups with 10 per group, i.e. normal control,positive control and three dose groups at 2.48 mg/kg, 4.96 mg/kg and9.92 mg/kg, respectively. They were fasted for 24 hours before theexperiment. The mice in the positive control group were given aninjection of 0.1 ml/10 g body weight of Neostigmine hypodermically whilethe mice in normal control groups were given rectal administrations ofsaline of 0.02 ml/10 g body weight. The mice in three dose groups weregiven rectal administrations of the Muskmelon-Base extract (diluted withsaline to desired doses and given 0.02 ml/10 g body weight). Twentyminutes after the medication 10% India ink (diluted with saline) wasgiven to each mouse at the dose of 0.2 ml/10 g body weight.

The mice were killed in 15 minutes and laparotomy was performed. Thesection of the small intestine from the pylorus to the ileocolicjunction was taken out and measured. The impelling percentage wascalculated by measuring the whole length of the small intestine and thedistance between the pylorus to the front of the ink impelled. (See FIG.3 for the result).

The two dose groups (intermediate and high dose) clearly showed smallintestine impelling effect in the experiment (p<0.01) by comparison withthe normal control group.

Experiment 5—Study on Large Intestine Impelling

This is an efficacy study on the large intestine movement in normal ratsafter rectal administration of the product.

The experimental result showed that the extract of the inventionpromotes significant large intestine impelling in rats.

In the experiment, 50 male Wistar rats weighing 200-250 g respectivelywere divided randomly into 5 groups with 10 per group. They were fastedfor 24 hours before the experiment. The rats were etherized in thesupine position. A midline incision (1.5-2 cm) was made at thehypogastrium level and the ileocolic junction was taken out delicatelyusing forceps. 10% India ink at 0.2 ml/100 g body weight withMuskmelon-Base extract at the dose of 1.5 mg/kg, 3.0 mg/kg or 6.0 mg/kgwas injected into the colon using a syringe. The rats in the normalcontrol and the positive control groups were given injections of salineand 10% ink. The rats in positive control were also given Neostigmineinjection (0.08 mg/kg). The incision was sewn up immediately after theinjections and a time count was started. The rats were killed andlaparotomy was performed 40 minutes after medication. The largeintestine from the appendix to the anus was taken out. The total lengthof the intestine was measured from the start of the colon to the anus.The distance of ink impulsion from the start of the colon to theink-front was also measured. The impelling percentage was calculated(FIG. 4).

By comparison with the normal control group, the extract at three doselevel all exhibited remarkable impelling effect on the large intestinein rats (p<0.01). Some dose-effect relation was observed.

Experiment 6—Gastric Emptying Experiment

In the experiment, 60 male ICR mice weighting 22-26 g respectively weredivided randomly into 5 groups with 12 per group, i.e. normal control,positive control and three dose groups, respectively. They were fastedfor 24 hours before the experiment. The mice in the positive controlgroup were given injection of 0.15 ml/10 g body weight of Neostigminehypodermic while the mice in normal control groups were given rectaladministrations of saline. The mice in three dose groups were givenrectal administrations of the Muskmelon-Base extract at 2.48 mg/kg, 4.96mg/kg and 9.92 mg/kg, respectively.

A suspension of 2% carboxymethyl cellulose with 0.05% phenol red wasgiven as gastric infusion to all the mice (0.4 ml/mouse) 30 minutesafter medication. The mice were killed and laparotomy was performed in15 minutes. Both ends of the stomach were tied up and the whole stomachincluding gastric content was cut into pieces and soaked in 5 ml of 1mol/L NaOH solution for 2 hours. It was centrifuged at 3000 r/min for 5minutes. 2.5 ml of the supernatant fluid was taken and 2.5 ml of 10%Trichloroacetic acid was added for albumen precipitation. It wascentrifuged again at 3000 r/min for 15 mins. and the supernatant fluidwas taken and analyzed with a spectrophotometer at 546 nm.

The percentage of gastric emptying was calculated using the followingformula and the results were compared with that of the normal controlgroup.

Percentage of the gastric emptying (Se)=100−Ps*Pa−1*100

-   -   Ps=phenol red concentration in the mouse stomach (ug/ml)    -   Pa=phenol red concentration after adding the same volume of 1        mol/L NaOH and Trichloroacetic acid (ug/ml) into the original        extract solution

By comparison with the normal control group (as shown in FIG. 5), thehigh-dose group (p<0.01) and the intermediate-dose group (p<0.05) showeda gastric emptying promoting effect with certain dose dependentfeatures.

Experiment 7—Study on Intestine Enterokinesia in Mice with MechanicalInjury

In this experiment, 72 male ICR mice weighing 22-26 g each were dividedrandomly into 6 groups with 12 per group. They were named as thepseudo-operation group, the model group, the positive control group(using Neostigmine) and 3 dose groups (low, intermediate and high dose).The mice were fasted for 24 hours before the experiment. The mice in thepseudo-operation group were etherized and a hole was made on each oftheir abdomens which were sewn up immediately after the incision. Themice in the other groups were also etherized and a hole made in each oftheir abdomen. Then a half-curved blunt object was inserted into thehole and rotated 5 turns in the anti-clockwise direction to make asimulative mechanical injury and then the hole was sewn up. After thesurgery, rectal administrations of saline (0.02 ml/10 g bodyweight) weregiven to the pseudo-operation group and the model group, and Neostigmineinjections (0.15 mg/kg) was given to the positive control group at thedose 0.1 ml/10 g of body weight. The 3 dose groups were given differentdoses of the Muskmelon-Base extract at the doses of 2.48 mg/kg, 4.96mg/kg, and 9.92 mg/kg (diluted with saline), respectively.

After medication, intragastric administration of 10% ink was given tothe mice at the dose of 0.2 ml/10 g body weight. The mice were killedand laparotomy was performed in 140 minutes. The intestine from thepylorus to the anus was taken out to calculate the impelling percentageby measuring the whole length of the alimentary tract and the distancefrom the pylorus to the front of the ink impelled (see FIG. 6).

As shown in FIG. 6, the extract in the three dose groups couldsignificantly promote intestine enterokinesia in mice with mechanicalinjury caused by surgery (p<0.01), which indicated that the extractcould significantly promote the recovery of enterokinesia in testingmice.

Experiment 8—Study on the Intestine Enterokinesia in Mice withGastrointestinal Hypo-Function Induced by Atropine

In this experiment, 66 male ICR mice weighing 22-26 g respectively weredivided randomly into 6 groups with 11 per group. They were fasted for24 hours before the experiment. The 6 groups were named as:

Normal control group,

Model group,

Positive control group, and

The three dose groups.

The mice in the normal control group were only injected saline at 0.1ml/10 g and mice in all other groups were injected 0.25 mg/kg ofAtropine hypodermic at 0.1 ml/10 g body weight.

Twenty minutes after injection Muskmelon-Base extract (diluted with thesaline) was given to the mice at the doses of 2.48, 4.96 and 9.96 mg/kgby rectal administration and saline solution was given to the mice innormal, control and the model groups. Neostigmine hypodermic injection(0.15 mg/kg) was given to the mice in the positive control group at thedose 0.1 ml/10 g body weight. A further 20 minutes later, anintragastric administration of 10% ink was given to all the mice at thedose 0.2 ml/10 g body weight.

All the mice were killed and laparotomized in 90 minutes. The intestinefrom the pylorus to the anus was taken out to measure the whole lengthof the alimentary tract. The distance from the pylorus to the front theink impelled was measured to calculate the impelling percentage.

As shown in FIG. 7, Muskmelon-Base extract at all three doses couldpromote intestinal enterokinesia in mice with gastrointestinalhypo-function induced by atropine (p<0.01), which indicated that theextract could promote the recovery of the hypo-functioned animals. Theeffect in the high dose group was similar to that of Neostigmine butwith no obvious muscle spasm side effect which was always found in allmice in the positive control group.

Experiment 9—Study on Excretion Intervals and the Quantity of Feces

In this experiment, 66 male ICR mice weighing 22-26 g respectively weredivided randomly into 6 groups with 11 per group. They were fasted for24 hours before the experiment.

The 6 groups were named as:

Normal control group,

Model group,

Positive control group, and

The three dose groups.

The mice in the normal control group were only injected saline at 0.1ml/10 g and mice in all other groups were injected 0.25 mg/kg ofAtropine hypodermic at 0.1 ml/10 g body weight.

Twenty minutes after injection Muskmelon-Base extract (diluted with thesaline) was given to the mice at the doses of 2.48, 4.96 and 9.96 mg/kgby rectal administration and saline solution was given to the mice innormal control and the model groups, Neostigmine hypodermic injection(0.15 mg/kg) was given to the mice in positive control group at the dose0.1 ml/10 g body weight.

A further 20 minutes later, an intragastric administration of 10% inkwas given to all the mice at the dose 0.2 ml/10 g body weight. Eachmouse was then individually caged in a box with white filter paper onfloor. The quantity of the feces and the time of excretion were recordedduring 4-hour observation periods.

Compared with the model group, the extract with total Cucurbitacins atthe low-dose (p<0.05), intermediate-dose (p<0.01) and high-dose (p<0.01)all significantly shortened excretion intervals in mice withgastrointestinal hypo-function and also increased the quantity of themice's feces (within 4 hour period). It showed that the extract canactivate the intestine enterokinesia restoration in mice withgastrointestinal hypo-function induced by atropine.

Experiment 10—Study on Proximal Colon Constriction in Anesthetized Rats.

In the experiment, 30 male Wistar rats weighing 300-350 g respectivelywere fasted for 24 hours before the experiment. The rats wereanesthetized with 10% Ethyl Carbamate in the supine position on aheat-insulating operating table. The hair on the middle abdomen was cutoff and a midline incision (5-6 μm) at the hypogastrium level was made.The colon and ileocolic junction was lifted out delicately using forcepsand a colon segment of about 2 cm from the ileocolic junction wasselected. Both ends of the segment (the ipsilateral colon tract) wereconnected by a suture to a fixed tube sheet. Another longer suture wasused, with one end connected to the colon wall, while the other end,through the tube, was connected to a JZ100 Muscle Tension Transducer(load 2.0 g). The colon segment was put back into the abdominal cavity,and the incision was sutured with the tube sticking out of the abdominalwall to prevent the colon segment from slipping. A piece of gauzesaturated with 0.9% sodium chloride solution was covered on theincision. After surgery, the RM6240 electrophysiology system wasconnected to record an enterogram.

After the enterokinesia was back to normal an enterogram was startedrecording. The rectal administration of Muskmelon-base extract was givenat the dose of 3.05 mg/kg and 6.10 mg/kg, respectively and theenterogram was recorded for further 2 hours. The following werecalculated:

-   -   The number of contraction waves in every 30 minutes (FIG. 8).    -   Amplitude index in every 30 minutes, i.e. total amplitude of all        the contraction waves in 30 minutes (FIG. 10).    -   The percentage of the change of the contraction wave (100% set        as before medication). It is calculated as follows: the number        of contraction waves in 30 mins. after medication−the number of        contraction waves in 30 mins. before medication/the number of        contraction waves in 30 mins. before medication×100% (FIG. 9).    -   The percentage rate of the change of amplitude indices (100% set        as before medication). It is calculated as follows: amplitude        index in 30 mins. after medication−amplitude index in 30 mins.        before medication)/amplitude index in 30 mins. before        medication×100%. (FIG. 11).

From the resulting data (FIGS. 8-11), Muskmelon-Base extract at both lowand high doses could:

Increase proximal colon movement in anesthetized rats.

It significantly increased

-   -   Contraction waves,    -   The amplitude indices,    -   The percentage of contraction numbers, and    -   The percentage of the amplitude index within 30 minutes. The        effect reached a peak between 30 minutes and 60 minutes. A        dose-effect relation was also observed.

Experiment 11—Study on Gastric Emptying

In this experiment, 60 male ICR mice weighing 22-26 g respectively weredivided into 6 groups with 10 per group. They were fasted for 24 hoursbefore the experiment.

The 6 groups included:

-   -   A normal control group,    -   A model group,    -   A positive control group (using Neostigmine), and    -   Three dose groups.

The mice in the normal, control group were given saline injectionswhilst the mice in the other 5 groups were given 0.3 mg/kg of atropinehypodermic injections.

The rectal administrations of saline were given to the normal controland the model group. The Muskmelon-Base extract (diluted with saline)was given to the mice in dose groups at 2.48 mg, 4.96 mg and 9.96 mg/kg,respectively.

The mice in positive control were given Neostigmine hypodermicinjections (0.15 mg/kg).

Intragastric administration of 0.05% phenol red and 2% carboxymethylcellulose were given to all the mice at 0.4 ml per mouse 30 minutesafter the medication. After 40 minutes the mice were killed andlaparotomized. Both ends of the stomach were tied up and the wholestomach including gastric content was cut into pieces and soaked in 8 mlof 1 mol/L NaOH solution for 2 hours. Then it was centrifuged at 3000r/min for 5 minutes and 2.5 ml of the supernatant fluid was taken. 2.5ml of 10% Trichloroacetic acid was added for albumen precipitation andthe solution was centrifuged again at 3000 r/min for 15 mins. Thesupernatant fluid was taken and analyzed with a spectrophotometer at 546nm. The percentage of gastric emptying was calculated by the followingformula.

Percentage of the gastric emptying (Se)=100−Ps*Pa−1*100

-   -   Ps=phenol red concentration in the mouse stomach (ug/ml)    -   Pa=phenol red concentration in the solution of the original        extract solution plus the same volume of 1 mol/L NaOH, and        Trichloroacetic acid (ug/ml)

As shown in FIG. 12, the extract with total Cucurbitacins at the highdose (9.96 mg/kg) could significantly improve the gastric emptyingfunction of the mice with gastrointestinal hypo-function induced byatropine (P<0.05). The differences in the low and intermediate dosegroups showed no significance (P>0.05) compared with the model groupalthough it showed trend towards promotion of gastric emptying effect.

Experiment 12 Study on Ex Vivo Guinea Pig Ileum (FIG. 13)

In this experiment, healthy, white, non-pregnant female guinea pigs,with a body weight of between 250-350 g were used. The Muskmelon_Baseextract was dissolved in 0.9% sodium chloride solution to the requiredconcentrations.

The guinea pigs were sacrificed after 24 hours fasting; the ileum wasremoved quickly and put into Tyrode's solution saturated with 5% CO2.After a normal cleansing process, it was cut to 1-2 cm and placed in aMagnus bath filled with Tyrode's solution for the measurement of musclemovement using a RM6240 multi-track physiological recorder.

After preparation of isolated ileum, the ileum tension was adjusted tonormalize the contraction and the normal movement was recorded using aRM6240 multi-track physiological recorder. 20 ml of test solution wasadded containing 30 mg, 60 mg and 120 mg extract, respectively and themovement recorded separately. Before the addition of 2^(nd) and 3^(rd)test solutions, the ileum was washed 2-3 times with 37 degree C.Tyrode's solution to normalize the ileum movement.

As shown in FIG. 13, the extract showed significant effect on increasingthe contracting strength of isolated guinea pig ileum.

1. A standardized extract of Cucumis melo L.
 2. The standardized extractof Cucumis melo L. as claimed in claim 1 comprising Cucurbitacin D andat least one of the following: Cucurbitacin A, Cueurbitacin B,Cucurbitacin E, Isocucurbitacin B and Isocucurbitacin D.
 3. Thestandardized extract of Cucumis melo L. as claimed in claim 1 comprisingCueurbitacin D and Cucurbitacin B.
 4. The standardized extract ofCucumis melo L as claimed in claim 1 which is a whole extract ofCucurbitacins.
 5. The standardized extract of Cucumis melo L. as claimedin claim 1 having a ratio of Cucurbitacin B to Cucurbitacin D of from30:1 to 1:2.
 6. The standardized extract of Cucumis melo L. as claimedin claim 5 having a ratio of Cucurbitacin B to Cucurbitacin D of from3:1 to 1:1.
 7. The standardized extract of Cucumis melo L. as claimed inclaim 1 standardized against Cucurbitacin D.
 8. The standardized extractof Cucumis melo L. as claimed in claim 1 standardized againstCucurbitacin B.
 9. The standardized extract of Cucumis melo L. asclaimed in claim 1 which is an aqueous extract.
 10. The standardizedextract of Cucumis melo L. as claimed in claim 9 having a an HPLCfingerprint substantially as depicted in FIG. 2 a as regards to thecompounds Cucurbitacin A peak 1, Cucurbitacin D peak 2, IsocucurbitacinD peak 3, Cucurbitacin B peak 4, Cucurbitacin E peak 5 andIsoucurbitacin B peak
 6. 11. The standardized extract of Cucumis melo L.as claimed in claim 1 which is an aqueous extract which has undergone analcohol precipitation.
 12. The standardized extract of Cucumis melo L.as claimed in claim 11 having a HPLC fingerprint substantially asdepicted in FIG. 2 c as regards to the compounds Cucurbitacin A peak 1,Cucurbitacin D peak 2, Isocucurbitacin D peak 3, Cucurbitacin B peak 4,Cucurbitacin E peak 5 and Isoucurbitacin B peak
 6. 13. The standardizedextract of Cucumis melo L. as claimed in claim 1 which is a chloroformpurified aqueous extract.
 14. The standardized extract of Cucumis meloL. as claimed in claim 13 having a an HPLC fingerprint substantially asdepicted in FIG. 2 d as regards to the compounds Cucurbitacin A peak 1,Cucurbitacin D peak 2, Isocucurbitacin D peak 3, Cucurbitacin B peak 4,Cucurbitacin E peak 5 and Isoucurbitacin B peak
 6. 15. The standardizedextract of Cucumis melo L. as claimed in claim 1 which is an alcoholicextract.
 16. The standardized extract of Cucumis melo L. as claimed inclaim 15 having a an HPLC fingerprint substantially as depicted in FIG.2 b as regards to the compounds Cucurbitacin A peak 1, Cucurbitacin Dpeak 2, Isocucurbitacin D peak 3, Cucurbitacin B peak 4, Cucurbitacin Epeak 5 and Isoucurbitacin B peak
 6. 17. The standardized extract ofCucumis melo L. as claimed in claim 1 which has undergone a resinpurification step.
 18. The standardized extract of Cucumis melo L. asclaimed in claim 17 having a an HPLC fingerprint substantially asdepicted in FIG. 2 e as regards to the compounds Cucurbitacin A peak 1,Cucurbitacin D peak 2, Isocucurbitacin D peak 3, Cucurbitacin B peak 4,Cucurbitacin E peak 5 and Isoucurbitacin B peak
 6. 19. The standardizedextract of Cucumis melo L. as claimed in claim 1 wherein the amount ofCucurbitacin D present in the total weight of cucurbitacins, includingisocucurbitacins, comprises from 5% to 50%, more preferably 8% to 41%.20. A pharmaceutical formulation comprising a standardized extract ofCucumis melo L in unit dosage form.
 21. The pharmaceutical formulationas claimed in claim 20 which is standardized against Cucurbitacin D. 22.The pharmaceutical formulation as claimed in claim 21 comprisingCucurbitacin D in an amount to provide a daily dose containing from0.0040 mg to 40 mg of Curcurbitacin D.
 23. The pharmaceuticalformulation as claimed in claim 22 comprising Cucurbitacin D in anamount to provide a daily dose containing from 0.040 mg to 4 mg ofCurcurbitacin D
 24. The pharmaceutical formulation as claimed in claim23 comprising Cucurbitacin D in an amount to provide a daily dosecontaining from 0.1 mg to 1.6 mg of Curcurbitacin D
 25. Thepharmaceutical formulation as claimed in claim 20 comprisingCucurbitacin B in an amount to provide a daily dose containing from0.0064 to 64 mg Curcurbitacin B.
 26. The pharmaceutical formulation asclaimed in claim 25 comprising Cucurbitacin B in an amount to provide adaily dose containing from 0.064 to 6.4 mg Curcurbitacin B.
 27. Thepharmaceutical formulation as claimed in claim 26 comprisingCucurbitacin B in an amount to provide a daily dose containing from 0.16to 2.6 mg Curcurbitacin B.
 28. The pharmaceutical formulation as claimedin claim 20 having a ratio of Cucurbitacin B:Cucurbitacin D of from 3:1to 1:1.
 29. The pharmaceutical formulation as claimed in claim 20wherein the amount of Cucurbitacin D present in the total weight ofCucurbitacins, including Isocucurbitacins, comprises from 5% to 50%,more preferably 8% to 41%.
 30. The pharmaceutical formulation as claimedin claim 20 which is for rectal administration.
 31. The pharmaceuticalformulation as claimed in claim 30 which is a suppository.
 32. Astandardized extract of Cucumis melo L for use in the manufacture of amedicament for the treatment of, or to effect, one or more of thefollowing conditions: Activating gastroenterokinesia; Postoperativegastroenterokinesia restoration; Shortening the duration ofpostoperative flatus; Treatments of constipation Treating postoperativeintestinal adhesion; Treating Intestinal adhesion and ileus; Stimulatinggastrointestinal movement in non-surgery Impelling the large and smallintestines; Activating gastric emptying; Smoothes bowel movement; andShortening excretion intervals and increasing the quantity of faeces.33. The standardized extract as claimed in claim 32 which does notproduce muscle spasm as a side effect.
 34. A method of treatment of thehuman or animal body comprising administering an extract orpharmaceutical formulation of the invention to a patient.